Re: [Histonet] prolonged impregnation in Golgi-Cox (Kolb) staining

From:

"Olek Michalski"

Dear Peggy,
thank you very much for your answer. It is pity I found it so late
(something went wrong with my mailbox) because I have already gave up
"saving" mentioned material. In spite of this circumstance I find it worth
to discuss.
I have actually processed one of these brains and tried to wash it in many
changes of water but it did not give good result. I mean tissue is dark
brown/green - so I could not see anything in thick sections I had cut.
Cutting is another issue since the tissue is cracking badly despite
soaking in 30% sucrose. I thought about using glycerin instead but I doubt
it is worth trying.
You mentioned air as oxidizing agent but I paid attention to fill the
containers up to the cork. What could it be then? Some tissue-based
oxidant? Or that the small amount of oxygen which was trapped under the
containers cork? I had not expect such a massive oxidation since I used
prolonged impregnation with chromates for other Golgi (w/o mercuric
chloride) techniques and did not observe this effect.
Finally answering your question. Golgi techniques are rather rare because
they are non-specific (at least they are believed to be so). The procedure
causes about 1-5% percent of the whole population of neurons to be stained
but the stained cell is (almost) completely filled with crystals. So (if
you are lucky) you can do nice morphology of the whole dendritic tree. I
was actually thinking about MAP2 IHC but it is impossible to trace one
cell's processes in such intensely stained tissue. On the other hand I
would like to apply some iontophoretic intracellular injections but it is
still future and really more laborious.
Best regards
Olek Michalski
> I don't know if the tissue can be "saved", but try washing in water for a
> few hours then process as usual for this technique, and see what happens
>
> A lot of things are working against you. The dichromate has been
> oxidized,
> the mercuric chloride has released a lot of hydrochloric acid which has
> been
> chewing up the proteins. And, boy, do you have a lot of fixative
> cross-links
> from both the chomate and the mercuric that you normally wouldn't have.
>
> As for the green color, I think that's due to the normally orangish/red
> potassium dichromate Cr+6 being oxidized to green Cr+3 in an acidic
> environment. The oxidizer is the air in the container, and the acidic
> environment comes from hydrochloric acid being released from the mercuric
> chloride during cross-linking with the tissue.
>
> I have a couple of questions, for anyone in the Histonet community. This
> Golgi-Cox fixation procedure comes up a on occasion on Histonet, mostly
> from
> researchers. How do these labs dispose of the chemicals afterwards?
> Mercuric
> chloride cannot be dumped down any sink, and most (but not all)
> water/sewer
> treatment plants won't allow potassium dichromate to be disposed down the
> sink either. And how do researchers dispose of the water and alcohol and
> xylene in the processing afterwards, where mercuric chloride and
> potassium
> dichromate are being pulled out of the tissue into the processing
> solutions?
>
> Can't these tissues be fixed in formalin (or some other less toxic
> fixative
> than mercury and chromium), and then IHC procedures done, such as
> antibodies
> against GFAP or neurofilaments or NSE? I work in a hospital, and am just
> wondering why this Golgi-Cox procedure is needed by researchers, but
> doesn't
> seem to be needed by clinical histology laboratories.
>
> Peggy A. Wenk, HTL(ASCP)SLS
> William Beaumont Hospital
> Royal Oak, MI 48073
>
> -----Original Message-----
> From: histonet-bounces@lists.utsouthwestern.edu
> [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of Olek
> Michalski
> Sent: Thursday, December 13, 2007 3:31 PM
> To: Histonet
> Subject: [Histonet] prolonged impregnation in Golgi-Cox (Kolb) staining
>
>
> Dear Histonetters,
>
> I have some brains which were left in Golgi-Cox chromation solution
> (K2Cr2O7, KCrO3, HgCl2: about 1% of each) for over 6 months. I would
> really
> like to use this material even if staining would not be very clear.
> Do you have an idea how to process the tissue in this case? Any help
> would
> be appreciated (and I know, next time I will be more careful about where
> I
> leave my preparates).
> By the way, I have already cut one of these brains and I found the
> sections
> to be dark green. I have noticed this colour in my G-C preparates before
> but
> mainly on the surface. I am actually curious whether the tissue should be
> more green or more red when I cut it. I mean should I shorten the normal
> period of chromation (about 14-15 days) if I see the brain is getting
> green?
>
> Yours truly
> Olek Michalski
--
Laboratory of Neurobiology
of Development and Evolution
Nencki Institute of Experimental Biology
ul. Pasteura 3, 02-093 Warszawa, Poland
Tel. +48 22 5892268, Fax +48 22 8225342
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